A variety of different hosts including plants, algae, fungi, stramenopiles and yeast are being investigated as means for commercial polyunsaturated fatty acid [“PUFA”] production. Genetic engineering has demonstrated that the natural abilities of some hosts (even those natively limited to linoleic acid [“LA”; 18:2 omega-6] and α-linolenic acid [“ALA”; 18:3 omega-3] fatty acid production) can be substantially altered to result in high-level production of various long-chain omega-3/omega-6 PUFAs. Whether this is the result of natural abilities or recombinant technology, production of arachidonic acid [“ARA”; 20:4 omega-6], eicosapentaenoic acid [“EPA”; 20:5 omega-3], docosapentaenoic acid [“DPA”; 22:5 omega-3] and docosahexaenoic acid [“DHA”; 22:6 omega-3] may all require expression of a delta-9 elongase gene.
Characterized delta-9 elongases have the ability to convert LA to eicosadienoic acid [“EDA”; 20:2 omega-6], and ALA to eicosatrienoic acid [“ETrA”; 20:3 omega-3]. However, only a few delta-9 elongases have been identified. These include the delta-9 elongases from Isochrysis galbana [“IgD9e”] (SEQ ID NOs:1 and 2; PCT Publications No. WO 2002/077213, No. WO 2005/083093, No. WO 2005/012316 and No. WO 2004/057001; GenBank Accession No. AAL37626), Eutreptiella sp. CCMP389 [“E389D9e”] (SEQ ID NOs:3 and 4; U.S. Pat. No. 7,645,604), Euglena gracilis [“EgD9e”] (SEQ ID NOs:7 and 8; U.S. Pat. No. 7,645,604) and Euglena anabaena [“EaD9e”] (SEQ ID NOs:11 and 12; U.S. Pat. No. 7,794,701). Although U.S. Pat. No. 7,645,604 identified seven motifs that were conserved between and among EgD9e, E389D9e and IgD9e elongases, only a single study has been performed with IgD9e in attempt to identify important amino acid residues to delta-9 elongase functionality (Qi, B., et al., FEBS Lett., 547:137-139 (2003)). There are no crystal structures available from delta-9 elongases to guide genetic evolution of the protein and little is known about the relationship between delta-9 elongase sequence, structure and function. Despite this lack of knowledge, there remains a need for delta-9 elongase genes that are efficiently expressed with high enzyme activities in production host cells capable of making PUFAs.
New delta-9 elongase mutants having high activity that are well suited for integration into PUFA biosynthetic pathways in commercially useful host cells have been discovered. Surprisingly and unexpectedly, it was found that specific point mutations resulted in delta-9 elongase mutants whose enzymatic activity was from 96% to 145% of the wildtype enzyme, based on the conversion of LA to EDA.